B99-02 Integrated Medical-Engineering Development of an Ultrasensitive Colorimetric Sensing System: Enabling Rapid, Highly Sensitive Detection of Mycobacterium Tuberculosis Antigens in Non-Sputum Specimens and Validation of Clinical Diagnostic Efficacy for Tuberculosis

Abstract Rationale Tuberculosis (TB) remains a global health crisis, with traditional sputum-based tests frequently failing to diagnose dry cough patients, the elderly, and children—who cannot provide qualified samples. This creates an urgent unmet need for rapid, non-sputum assays. Serum is readily accessible and contains Mycobacterium tuberculosis (MTB)-specific antigens (immune status-independent), but current commercial methods lack adequate sensitivity for early detection. To address this gap, we developed a colorimetric ultrasensitive platform via medical-engineering integration, combining bimetallic nanozyme catalysis and magnetic-assisted enrichment, to enhance MTB antigen detection in non-sputum samples for accurate, early TB diagnosis. Methods A herringbone-shaped microfluidic channel was designed, with finite element analysis optimizing key parameters for uniform and efficient reactions. Silver-platinum bimetallic nanozymes were synthesized in situ in the microfluidic system; first-principles calculations clarified how heteronuclear metal doping regulates the nanozymes’ electronic structure and d-band center, guiding doping ratio optimization to enhance substrate adsorption, catalytic efficiency, and peroxidase-like activity. TB MPT64 antigen-specific aptamers were immobilized as signal probes, combined with magnetic nanoparticle-based capture probes to form a sandwich structure—integrating magnetic-assisted enrichment and colorimetric detection of target antigens. Ultraviolet spectroscopy verified nanozyme activity and characterized colorimetric signal responses; key sensor parameters (limit of detection [LOD], specificity) were determined, with preclinical studies compared against commercial methods. Results The bimetallic nanozymes had uniform particle size, good dispersibility, higher catalytic efficiency than natural peroxidases, strong environmental stability, and reliable signal output. The colorimetric system detected MPT64 antigen within 2 hours, with a linear range of 1 fg/mL to 10 ng/mL and a minimum LOD of 0.26 fg/mL—far lower than commercial and most of other reported TB antigen detection methods. It showed good reproducibility and high specificity. In preclinical small-sample testing (95 subjects: 70 TB patients, 25 non-TB patients), it had excellent diagnostic efficacy for active pulmonary TB, with an AUC of 0.935, sensitivity of 96.7%, and specificity of 95.3%. Conclusion Our medical-engineering integrated platform represents a transformative advance for non-sputum TB diagnosis. By enabling ultra-sensitive, rapid serum MPT64 antigen detection, it addresses the critical limitations of existing non-sputum methods and meets WHO Target Product Profile standards. With strong potential for point-of-care (POC) application in resource-limited settings, this platform could revolutionize early TB diagnosis and accurate identification—ultimately improving patient outcomes and supporting global TB elimination efforts. Further validation in large, multi-center cohorts is ongoing. This abstract is funded by: None